This project is aimed at understanding receptor-ligand interactions in the large family of olfactory odor receptors. The approach will develop a new high throughput expression system for expressing odor receptors in heterologous systems that are convenient for assaying hundreds to thousands of potential ligands for activity. In the first major aim we will identify and clone a hypothesized co-factor required for the proper targeting of odor receptors to the membrane. This factor is expected to be a small protein that accompanies odor receptor protein from the ER to the plasma membrane and may be involved in differential glycosylation of the mature protein. We will utilize a novel expression cloning strategy in which functional expression of a particular odor receptor is coupled to survival in specialized strains of yeast. The identification of a co-factor or co-factors important in receptor membrane targeting should allow us to develop a high throughput screen similar to other GPCRs. We will base this screen in yeast since these eukaryotic cells have only one endogenous GPCR, which can be knocked out, providing an unambiguous screening background. In addition we will institute a screening system in a mammalian cell line as well. The ability to screen large numbers of odor ligands on an extensive group of odor receptors will allow us to generate crucial data relating ligand selectivity and affinity to specific receptor sequences. Receptor sequences paired with cognate chemical ligands will be developed into a large database, the analysis of which will provide pharmacological data for odor receptors comparable to that for other GPCRs. Finally we will produce amino acid mutations in residues hypothesized to be influential in ligand binding. Results from these investigations will apply to structure-function issues in olfactory receptors specifically and GPCRs generally. Odor receptors comprise the largest family of G -protein. coupled receptors in the mammalian genome. As such they represent a kind of "natural" experiment in which variations in receptor structure due to differences in amino acid sequence can be related to alterations in receptor-ligand affinity. Because the odor receptors are in the same genetic superfamily of receptors as those for neurotransmitters, hormones, drugs and many peptides, understanding the relation between structure and function in these receptors could lead to important new principles for rational drug design and for receptor modifications. Many neurological diseases are the result of deficient receptors for neurotransmitters or neurohormones. These results will speak directly to the those causes.